Genetic and functional characterization of candidate genes for complex psychiatric diseases using next-generation sequencing and cellular uptake assays

Complex phenotypes are the result of a complex interplay between genes and environmental factors. Extensive linkage, candidate and genome-wide association studies (GWASs) have been carried out to unravel genetic risk variants for human diseases. The identification of genes, involved in the pathomechanism of a disease, might be beneficial for its diagnosis, treatment and prognosis. While GWASs allowed the identification of a large number of common variants robustly associated with common complex diseases, the heritability, which can be explained by these variants, is small. The discrepancy between the estimated heritability from twin, family and adoption studies and the heritability obtained from GWAS was termed ?missing heritability? and led to the investigation of additional factors that might also contribute to disease susceptibility, including gene-environment interactions, gene-gene interactions, structural variants and rare variants. In this thesis, the role of less common and rare variants in susceptibility to common complex diseases was investigated. In order to accomplish this, a candidate gene for panic disorder (PD) and a possible risk gene for major depressive disorder (MDD) were screened for the presence of common and rare variants using next-generation sequencing in a pooled approach. In a previously published GWAS, a haplotype containing two common intronic variants in the transmembrane protein 132D (TMEM132D) gene was associated with PD. Another GWAS identified solute carrier family 6 member 15 (SLC6A15), which encodes an amino acid transporter, as a risk gene for MDD. A common intergenic variant about 600 kilobase downstream of this gene was shown to decrease SLC6A15 gene expression in lymphoblastoid cell lines and hippocampus. Susceptibility genes for complex diseases, identified in GWAS, are promising candidates for the search of rare variants as genes harbouring common variants are likely to contain also rare variants. Pooled targeted re-sequencing of the exonic regions of TMEM132D in 300 anxiety disorder patients, mostly suffering from PD (84.7%), and 300 healthy controls allowed the detection of 371 genetic variants. Of these variants, 24.0% were common (minor allele frequency (MAF) \ensuremath> 5.0%), whereas the vast majority was less common (MAF 1.0 ? 5.0%) to rare. 247 variants had not been reported before, including 12 novel non-synonymous variants leading to an amino acid exchange in the protein. While common variants associated with PD were not identified, an overrepresentation of non-synonymous variants and variants with predicted changes on splicing in healthy controls compared to PD patients was observed. These putatively functional relevant variants were distributed along a broad MAF spectrum, ranging from 0.17 to 30.0%. In addition, a higher rate of private non-synonymous variants, which were only present in either cases or controls in this study, but not in over 7,500 individuals with different ethnic backgrounds from other publicly available re-sequencing datasets, in patients compared to controls was seen. Combined with the data from the previous GWAS study in which the association with PD was carried by common variants, this pooled re-sequencing study suggests that not only common or rare variants alone, but a combination of both contributes to the development of anxiety-related phenotypes. Re-sequencing the whole SLC6A15 locus in 400 MDD patients and 400 healthy controls, 405 genetic variants were identified, including twelve non-synonymous variants. Only 15.0% of the detected variants were common. While none of the non-synonymous variants was significantly associated with MDD, two rare non-synonymous variants were identified to influence protein function. In contrast to the TMEM132D protein whose molecular function has still to be discovered, SLC6A15 is known to transport neutral amino acids into predominantly neuronal cells. The cellular uptake of neutral amino acids such as proline is thus a measurable property that associates with function. The uptake experiments identified two rare variants to be associated with a significant increase in proline uptake in HEK cells. This result suggests that rare variants in SLC6A15 might influence the biochemical function of its amino acid transporter and thus downstream neuronal function and possibly the risk for MDD and other stress-related psychiatric disorders. In addition, this study highlights that functional exploration of genetic variants might be promising to identify putatively disease-relevant variants as statistically significant associations for rare variants might only be achieved in extremely large samples

Genetic and functional characterization of candidate genes for complex psychiatric diseases using next-generation sequencing and cellular uptake assays

Complex phenotypes are the result of a complex interplay between genes and environmental factors. Extensive linkage, candidate and genome-wide association studies (GWASs) have been carried out to unravel genetic risk variants for human diseases. The identification of genes, involved in the pathomechanism of a disease, might be beneficial for its diagnosis, treatment and prognosis. While GWASs allowed the identification of a large number of common variants robustly associated with common complex diseases, the heritability, which can be explained by these variants, is small. The discrepancy between the estimated heritability from twin, family and adoption studies and the heritability obtained from GWAS was termed “missing heritability” and led to the investigation of additional factors that might also contribute to disease susceptibility, including gene-environment interactions, gene-gene interactions, structural variants and rare variants. In this thesis, the role of less common and rare variants in susceptibility to common complex diseases was investigated. In order to accomplish this, a candidate gene for panic disorder (PD) and a possible risk gene for major depressive disorder (MDD) were screened for the presence of common and rare variants using next-generation sequencing in a pooled approach. In a previously published GWAS, a haplotype containing two common intronic variants in the transmembrane protein 132D (TMEM132D) gene was associated with PD. Another GWAS identified solute carrier family 6 member 15 (SLC6A15), which encodes an amino acid transporter, as a risk gene for MDD. A common intergenic variant about 600 kilobase downstream of this gene was shown to decrease SLC6A15 gene expression in lymphoblastoid cell lines and hippocampus. Susceptibility genes for complex diseases, identified in GWAS, are promising candidates for the search of rare variants as genes harbouring common variants are likely to contain also rare variants. Pooled targeted re-sequencing of the exonic regions of TMEM132D in 300 anxiety disorder patients, mostly suffering from PD (84.7%), and 300 healthy controls allowed the detection of 371 genetic variants. Of these variants, 24.0% were common (minor allele frequency (MAF) > 5.0%), whereas the vast majority was less common (MAF 1.0 – 5.0%) to rare. 247 variants had not been reported before, including 12 novel non-synonymous variants leading to an amino acid exchange in the protein. While common variants associated with PD were not identified, an overrepresentation of non-synonymous variants and variants with predicted changes on splicing in healthy controls compared to PD patients was observed. These putatively functional relevant variants were distributed along a broad MAF spectrum, ranging from 0.17 to 30.0%. In addition, a higher rate of private non-synonymous variants, which were only present in either cases or controls in this study, but not in over 7,500 individuals with different ethnic backgrounds from other publicly available re-sequencing datasets, in patients compared to controls was seen. Combined with the data from the previous GWAS study in which the association with PD was carried by common variants, this pooled re-sequencing study suggests that not only common or rare variants alone, but a combination of both contributes to the development of anxiety-related phenotypes. Re-sequencing the whole SLC6A15 locus in 400 MDD patients and 400 healthy controls, 405 genetic variants were identified, including twelve non-synonymous variants. Only 15.0% of the detected variants were common. While none of the non-synonymous variants was significantly associated with MDD, two rare non-synonymous variants were identified to influence protein function. In contrast to the TMEM132D protein whose molecular function has still to be discovered, SLC6A15 is known to transport neutral amino acids into predominantly neuronal cells. The cellular uptake of neutral amino acids such as proline is thus a measurable property that associates with function. The uptake experiments identified two rare variants to be associated with a significant increase in proline uptake in HEK cells. This result suggests that rare variants in SLC6A15 might influence the biochemical function of its amino acid transporter and thus downstream neuronal function and possibly the risk for MDD and other stress-related psychiatric disorders. In addition, this study highlights that functional exploration of genetic variants might be promising to identify putatively disease-relevant variants as statistically significant associations for rare variants might only be achieved in extremely large samples

Study of genetic factors in treatment-related complications in patients with childhood acute lymphoblastic leukemia and post transplantation of hematopoietic stem cells

La leucémie lymphoblastique aiguë (LLA) est le cancer le plus fréquent chez les enfants. Malgré le fait que plus de 80% des enfants atteints de LLA sont aujourd'hui guéris de leur maladie, ce succès a toutefois un prix élevé, car l’exposition aux médicaments cytotoxique et/ou à l’irradiation pendant une période vulnérable du développement de l’enfant peut entraîner des conséquences à long terme. En effet, environ 60% des enfants ayant survécu à une LLA devront vivre avec des problèmes de santé liés au traitement, également appelés effets indésirables tardifs (late-adverse effects, LAEs). Parmi ces derniers, on notera des problèmes métaboliques, l’ostéoporose, une altération des fonctions cognitives ou cardiaques, ainsi que la dépression et l’anxiété. Si certains survivants ne présentent aucune de ces complications, d'autres peuvent en avoir plusieurs. Différents facteurs peuvent contribuer à cette variabilité, notamment le traitement reçu, les caractéristiques de la maladie, les habitudes de vie et, surtout, la constitution génétique du patient. Ce projet s'est concentré sur les biomarqueurs génétiques permettant d'identifier les individus les plus susceptibles de souffrir de LAEs. Récemment, une étude exhaustive (évaluations cliniques, psychosociales et biochimiques) s’est déroulée au CHU Sainte-Justine pour caractériser chacune de ces morbidités chez 250 survivants de la LLA de l'enfant (cohorte PETALE). De plus, on a obtenu le profil génétique de chaque participant. Nous avons utilisé cet ensemble de données et des outils statistiques et bio-informatiques pour réaliser des études d'association comparant la fréquence des variants génétiques chez les survivants ayant développé ou non des LAEs; en particulier, les complications cardiovasculaires et neurocognitives, ainsi que les troubles de l'humeur tels que l'anxiété et la dépression. D'autres facteurs de risque tels que les caractéristiques de traitement et/ou de la leucémie ont été pris en compte lors de l'analyse pour dériver les meilleurs prédicteurs génétiques. Ainsi, en utilisant l'approche des gènes candidats, nous avons identifié les variants communs des gènes MTR, PPARA, ABCC3, CALML5, CACNB2 et PCDHB10 qui étaient associés à des déficits de performance des tests neurocognitifs, tandis que les variants des gènes SLCO1B1 et EPHA5 étaient associés à l'anxiété et à la dépression. Deux variants, rs1805087 dans le gène MTR et rs58225473 dans le gène CACNB2 sont particulièrement intéressants, car ces associations ont été validées dans la cohorte de réplication SJLIFE (St. Jude Children's Research Hospital, Memphis, USA). Les analyses d'association ont été complémentées par une étude d'association à l'échelle de l'exome, qui a identifié plusieurs gènes supplémentaires comme des modulateurs potentiels du risque de développer des complications neurocognitives liées au traitement (gènes AK8 et ZNF382), ainsi que l'anxiété et la dépression (gènes PTPRZ1, MUC16, TNRC6C-AS1, APOL2, C6orf165, EXO5, CYP2W1 et PCMTD1). Le variant rs61732180 du gène ZNF382 a ensuite été validé dans la cohorte de réplication SJLIFE. Également, nous avons effectué des analyses d’association concernant les complications cardiaques liées au traitement qui ont identifié plusieurs nouveaux marqueurs associés à ces complications dans les gènes TTN, NOS1, ABCG2, CBR1, ABCC5, AKR1C3, NOD2 et ZNF267. De plus, nous avons résumé les connaissances actuelles sur les marqueurs pharmacogénomiques qui ont été associés aux effets de cardiotoxicités, induites par les anthracyclines, qui affectent les patients atteints de cancer pédiatrique. Nous avons également inclus un aperçu de l'applicabilité des résultats rapportés, notamment ceux qui ont été validés dans la cohorte PETALE. Par ailleurs, nous nous sommes intéressés aux complications qui surviennent après une greffe de cellules souches hématopoïétiques. Nous avons appliqué des approches bio-informatiques et statistiques similaires pour obtenir un profil plus complet de la composante génétique derrière ces complications potentiellement mortelles. Ainsi, une étude d'association à l'échelle de l'exome a été réalisée dans une cohorte de patients pédiatriques subissant une greffe de cellules souches hématopoïétiques après un régime de conditionnement contenant du busulfan. Nous avons identifié de nouvelles variations génétiques conférant un risque plus élevé de syndrome d'obstruction sinusoïdale (notamment dans les gènes UGT2B10, BHLHE22, et KIAA1715) et de maladie aiguë du greffon contre l'hôte (dans les gènes ERC1, PLEK, NOP9 et SPRED1), qui pourraient être utiles pour des stratégies personnalisées de prévention et de traitement. Ces travaux contribuent à la compréhension de l'influence des facteurs génétiques sur le risque de développer des complications liées au traitement, tant au cours du traitement qu'à long terme. De plus, les marqueurs génétiques signalés ainsi que d'autres facteurs de risque connus peuvent conduire à des modèles de prédiction identifiant les patients à risque accru de ces complications.Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Even though more than 80% of children with ALL are now cured of their disease, this success comes at a high price as exposure to cytotoxic drugs and/or radiation during a vulnerable period of child development may have long-term consequences. In fact, approximately 60% of children who survive ALL will have to live with treatment-related health problems, also called late-adverse effects (LAEs). These include metabolic problems, osteoporosis, impaired cardiac or cognitive functions, as well as depression and anxiety. While some survivors do not have any of these complications, others may have more than one. Different factors can contribute to this variability, in particular, the treatment received, the characteristics of the disease, the lifestyle, and, above all, the genetic makeup of the patient. This project focused on genetic biomarkers capable of identifying the individuals most likely to suffer from LAEs. Recently, an exhaustive study (clinical, psychosocial, and biochemical evaluations) took place at Sainte-Justine University Health Center (Montreal, Canada), with the goal to characterize each of these morbidities in 250 survivors of childhood ALL (PETALE cohort). In addition, the genetic profile of each participant was obtained, and we used statistical and bioinformatics tools to perform association studies on this dataset in order to compare the frequency of genetic variants in survivors with or without LAEs. We evaluated cardiovascular and neurocognitive complications, as well as mood disorders such as anxiety and depression. Other risk factors, such as treatment and/or leukemia characteristics were also considered during the analysis to derive the best genetic predictors. Thus, using the candidate gene approach, we identified common variants in the MTR, PPARA, ABCC3, CALML5, CACNB2, and PCDHB10 genes that were associated with deficits in neurocognitive tests performance, whereas variants in the SLCO1B1 and EPHA5 genes were associated with anxiety and depression. Two variants, rs1805087 in the MTR gene and rs58225473 in the CACNB2 gene, are of particular interest since these associations were validated in an independent SJLIFE replication cohort (St. Jude Children's Research Hospital, Memphis, USA). The association analyses were complemented by an exome-wide association study, which identified several additional genes as potential modulators of the risk of developing treatment-related neurocognitive complications (genes AK8 and ZNF382), as well as anxiety and depression (genes PTPRZ1, MUC16, TNRC6C-AS1, APOL2, C6orf165, EXO5, CYP2W1, and PCMTD1). Variant rs61732180 in the ZNF382 gene was further validated in the replication SJLIFE cohort. To a great extent, we performed association analyses regarding treatment-related cardiac complications which identified several novel markers associated with these toxicities in the TTN, NOS1, ABCG2, CBR1, ABCC5, AKR1C3, NOD2, and ZNF267 genes in survivors of childhood ALL. In addition, we summarized the current knowledge on pharmacogenomic markers related to anthracycline-induced cardiotoxicity affecting pediatric cancer patients. We also included a brief overview of the applicability of reported findings to the PETALE cohort, validating several of them. Besides, we were interested in the complications that arise after a hematopoietic stem cell transplantation. We applied similar bioinformatics and statistical approaches to gain a more complete insight into the genetic component behind these life-threatening complications. Thus, an exome-wide association study was performed in a cohort of pediatric patients undergoing hematopoietic stem cell transplantation following a conditioning regimen containing busulfan. Our results identified new genetic variations conferring a higher risk of sinusoidal obstruction syndrome (notably in the UGT2B10, BHLHE22, and KIAA1715 genes) and acute graft-versus-host disease (ERC1, PLEK, NOP9, and SPRED1 genes), which could be useful for personalized prevention and treatment strategies. This work contributes to the understanding of the influence of genetic factors on the risk of developing treatment-related complications, both during treatment and in the long term. Furthermore, the reported genetic markers along with other known risk factors can lead to prediction models identifying patients at increased risk for these complications

Assessment of Genome-Wide Genetic and Epigenetic De Novo Variation in Families with Monozygotic Twins Discordant for Schizophrenia

Schizophrenia (OMIM: 181500) is a common, debilitating and life-altering disorder. It affects 1% of the population worldwide and most often presents in early adulthood leading to devastating effects for patients, their families and society. Despite thousands of studies performed on the underlying mechanisms of schizophrenia, the causes of the disease remain unknown. However, what is known is that environmental, genetic and epigenetic factors contribute to the development of this complex disorder. Although a genetic role in schizophrenia is well established, the search for schizophrenia genes using traditional approaches has remained challenging. Interestingly, monozygotic twins show concordance for schizophrenia only 50% of the time and therefore provide a unique scenario for genomic analysis. This Doctoral thesis examines the genetic and epigenetic contributions to schizophrenia discordance in monozygotic twins. In this thesis, I have identified and characterized genome-wide changes through the use of the Affymetrix SNP 6.0 Microarray, Complete Genomics whole genome sequencing and the Nimblegen Methylation 720k Microarray. Specifically, I have identified genetic and epigenetic differences between monozygotic twins discordant for schizophrenia. The results show multiple genetic and epigenetic changes between monozygotic twins with discordance for schizophrenia. Some of these differences are patient-specific and others are shared between affected twins in the study. In addition, some of these differences affected genes and others did not. Many of the genes and genomic regions have been previously implicated in schizophrenia and neurodevelopmental disorders. The findings reinforce the concept that individual genomes harbor extensive variability, some inherited and some acquired. Even monozygotic twins are not identical and each individual may be a mosaic; carrying different sequence variations in different cells. The results also suggest that discordance for schizophrenia in monozygotic twins may result from the accumulation of genetic and epigenetic mutations that lead to the disease threshold being met in one twin only. The results argue for the involvement of de novo mutations in genetic individuality and complex disease. Improved understanding of the genomic contributions to schizophrenia is critical for movement towards earlier and more accurate diagnosis, better treatment and further understanding of this complex mental health disorder

Busca de variantes raras en xenes asociados á esquizofrenia

A esquizofrenia é unha enfermidade multifactorial complexa xa que na súa aparición interveñen tanto factores ambientais como xenéticos. Esta tése toma de referencia a hipótese enfermidade común- variante rara que sostén que existen múltiples variantes pouco frecuentes na poboación, que teñen un risco asociado moderado e que estarían influíndo no desenvolvemento da esquizofrenia. Nesta tese tratabamos de buscar variantes raras funcionais en DISC1, un xene asociado á doenza, onde viamos que existía unha posible variante funcional asociada a enfermidade na rexión rexión 3´UTR do xene. A continuación estudabamos o papel das variantes comúns non sinónimas no interactoma de DISC1 atopando unha sobre-representación das proteínas que forman parte do interactoma de DISC1, sinalando a este conxunto de xenes como un factor a ter en conta no risco a padecer esquizofrenia. Noutro dos traballo, mediante a secuenciación de nova xeración estudabamos variantes raras cun efecto alto en 21 xenes pertencentes ó interactoma de DISC1 ou que previamente foran asociados á esquizofrenia mediante estudos de GWAS ou CNVs. Así atopabamos unha variante cunha significación nominal presente no xene AKAP9, ademais dunha asociación nominal nos xenes ZNF804A e NRXN1despois de levar a cabo unha análise de acumulación de variantes. Por último, secuenciabamos NRXN1 no total da mostra e levabamos acabo unha meta-análise confirmando un exceso deste tipo de variantes en controis